Thermo Fisher Scientific MASS Owner's manual

Brand: Thermo Fisher Scientific

Model: MASS

Type: Owner's manual

This tutorial describes how to detect structurally-related compounds by running a Fragment Ion Search™

(FISh) analysis. Applying a FISh filter to a chromatogram removes all the spectral peaks except for the

defined FISh model peaks. You can use this feature to extract spectral peaks for structurally-related

features, for example, compounds related to the parent drug in metabolite ID, API impurity ID, or

compounds that share a common list of fragments.

Contents

•Demo data files

• Check the global settings

• Run and Review a FISh analysis

• Identify detected components

• Save the results to an HCCX file

• Create and edit a fragments list

Demo data files This tutorial uses the following files that reside in the Demo Data folder on the application computer.

Check the

global settings

Before you begin this tutorial, check the global settings for the mass accuracy and color-coding of the

spectrum peaks.

YTo restore the default settings for mass accuracy and spectrum peak color-coding

1. Open the Mass Frontier application by double-clicking its desktop icon, , or by choosing

Thermo Mass Frontier 8.1 > Mass Frontier 8.1 from the Windows™ Start menu.

The application opens to the Mass Frontier startup window or the Modules & Tools toolbar.

Mass Frontier 8.1 tutorial to Detect Structurally-Related

Compounds with the FISh Algorithm

File Description

OMP-Sigma-70K. raw A raw data file acquired with an LC-ESI/MS/MS experiment using

an Orbitrap™ QExactive™ Plus MS. The sample solution was

prepared from an omeprazole standard. Omeprazole is a therapeutic

drug for acid reflux.

Omeprazole FISh Fragments.sdf A structure file that contains twelve fragment structures for the

parent compound—omeprazole

OMP-Sigma-70K.chpro.jcd A component detection file that contains the component detection

settings for an LC/MS experiment

Omeprazole.mol A structure file that contains the structure for omeprazole—the

analyte of interest in this tutorial

Revision A B51001048

2. Do one of the following:

• From the Mass Frontier startup window, click Global Settings.

Figure 1. Mass Frontier startup window

–or–

• From the application tab bar, click the Start tab to display the Start menu, and then choose

Global Settings.

Note If you clear the Show this Window Next Time check box, the next time you open the

application, it opens with only the Modules & Tools toolbar displayed.

Click to open the Global Settings dialog box.

The Global Settings dialog box opens (Figure 2).

3. In the left pane, under Layout, click MS Spectrum, and then click Restore Default.

Figure 2. Global Settings dialog box – MS Spectrum view with the default settings

4. In the left pane under Parameters, click Mass Tolerance, and then click Restore Default.

Start tab

Start menu

Run and Review

a FISh analysis

To run a FISh analysis and review the results, follow these topics in order:

1. Open a raw data file for processing

2. Apply a fragment ion search filter

3. View the FISh trace

4. Review the detected components

Open a raw data

file for

processing

YTo open the example raw data file

1. In the Modules & Tools toolbar, click Chromatogram Processor.

IMPORTANT Make sure that the Determine from Source option (default) is selected for the mass

accuracy of the experimental data; a user-defined setting for mass accuracy can change the

number of detected components.

Figure 3. Modules & Tools toolbar

2. In the Open Chromatogram dialog box, browse to the following folder, select OMP-Sigma-70K.raw,

and click Open.

drive:\Users\Public\Public Documents\HighChem\Mass Frontier 8.1\Demo

Data\Chromatograms

A new instance of the Chromatogram Processor module opens as a tabbed document with the

following views (Figure 4):

• The chromatogram data view at the upper left lists the scan data by scan level and number.

• The chromatogram view at the upper right displays the total ion current (TIC) chromatogram.

The y-axis scale is set to absolute intensity.

•The MS spectrum view at the lower right displays the first scan in the raw data file.

• The command processor view at the lower left is empty until you apply actions to the

chromatogram.

Note Applying a component detection algorithm to the chromatogram adds a list of detected

components to this pane.

Note To change the scale from absolute counts to relative intensity (versus the base mass

spectrum peak), right-click the view and choose Show Absolute Intensities.

Application tab bar

Modules & Tools toolbar

Figure 4. TIC chromatogram and scan number 1 for the selected raw data file

Apply a fragment

ion search filter

YTo apply a fragment ion search filter to the TIC chromatogram

1. In the Actions group of the Chromatogram Processor toolbar, click FISh.

The Model page of the FISh Detection view opens to the right of the chromatogram and spectrum

views (Figure 5).

Note Large data files can take a significant time to load. The status bar at the bottom of the

application window provides information about the loading progress, from reading the scan data

to building the scan tree.

Tip To show or hide the views on a Chromatogram Processor page, click the following icons in the View area

of the Chromatogram Processor toolbar:

• For the MS spectrum view, click the Show MS Spectrum icon, .

• For the chromatogram data view, click the Show Chromatogram Data icon, .

• For the command processor view, click the Show Command Processor icon, .

You cannot hide the chromatogram view.

TIC page of the

chromatogram view

Spectrum page of the

MS spectrum view

Chromatogram data

view

Command

processor view

Note The fragment ion search filter that you specify for FISh detection is called the FISh model.

Note You use the Model page to set up the FISh model for the fragment ion search.

FISh button

Figure 5. Model page of the FISh Detection view with the default settings

2. In the Fragments area, set up the fragments list as follows:

a. Select the Fragments option.

The FISh Filter: Input Fragments dialog box opens.

b. Click the Import icon, , and then click Import File.

c. Browse to the following folder, select Omeprazole FISh Fragments.sdf, and click Open.

drive:\Users\Public\Public Documents\HighChem\Mass Frontier 8.1\Demo Data\Structures

Twelve structures appear in the dialog box (Figure 6). The structure with the highest m/z value

(m/z 346.12199) is the protonated ion of omeprazole.

Tip To edit the fragment options after closing the FISh Filter: Input Fragments dialog box,

click Edit.

Restores the default

parameter settings

on the Options page

Fragments option—

Opens the FISh

Filter:Input Fragments

dialog box

Figure 6. Fragment structures in the Omeprazole FISh Fragments.sdf file

d. Click OK.

The number of fragments to use appears to the right of the Fragments option.

3. Add one modification to the FISh model as follows:

a. On the Model page of the FISh Detection view, select the Modifications check box.

The FISh Modifications dialog box opens.

b. In the Predefined Modifications area, select the Oxidation (+O) check box, and then click Add

(Figure 7).

Note For a FISh analysis, make sure that the fragments list includes the analyte’s precursor

ion.

Tip To edit the FISh modifications after closing the FISh Modifications dialog box, click

Edit.

Precursor ion for the

analyte of interest

Number of fragments

Figure 7. FISh Modifications dialog box with one modification

c. Click OK.

The number of modifications to consider appears to

the right of the Modifications check box.

4. To reset the parameter settings on the Options page, click the Reset icon, .

5. Click the Options tab to open the Options page (Figure 8). Keep the default settings for selecting

which spectral peaks contribute to the ion current at each time point in the FISh chromatogram.

Figure 8. Options page of the FISh Detection view

Note You use the Options page to specify the component detection algorithm and which spectral

peaks you want the application to use to build the FISh chromatogram.

Number of

modifications

Opens the JCD

dialog box.

Identifies the

spectral peaks

that correspond

to the FISh model

fragments.

6. Set up component detection as follows:

a. On the Options page of the FISh Detection view, click Options.

The JCD dialog box opens (Figure 9).

By default, all the sliders in the Deconvolution pane of the JCD view are set to the middle.

b. In the JCD dialog box, click Load.

c. Browse to the following folder, select the OMP-Sigma-70K.chpro.jcd file, and click Open.

drive:\Users\Public\Public Documents\HighChem\Mass Frontier 8.1\Demo

Data\Chromatograms

The selected .chpro.jcd file moves the Intensity of Detected Component slider to the left, which

increases the minimum intensity threshold for a component and typically decreases the number

of detected components. It also slightly changes the Component Overlapping Intensity setting.

Figure 9. Component detection settings (from selected file)

d. Click OK.

7. In the FISh Detection view, click Preview.

8. After the analysis finishes, click Accept.

Modified settings

The command processor view lists the FISh Detection filter as an applied action. A list of detected

components appears in the chromatogram data view, and the blue triangles indicate the detected

components.

Figure 10. Accepted FISh analysis

View the FISh

trace Use the options menu to display selected chromatograms.

YTo view the FISh chromatogram

Open the options menu and clear the Show TIC, Show All Components, and

Show Selected Components check boxes.

A FISh chromatogram is a TIC chromatogram that is calculated from the

identified (marked) m/z peaks for the specified FISh model. By default, the application displays

calculated TIC chromatograms in pink (Figure 11).

Applied actions in the

command processor view

Number of detected components in the

chromatogram data view—12

Tip You can change the color settings for chromatogram in the 2D Chromatogram view of the

Global Settings dialog box.

Figure 11. FISh chromatogram (marked peaks chromatogram)

Review the

detected

components

YTo inspect the spectra for a detected component

1. In the Components list, select Component 5 (m/z 362.1166 at 9.036 min).

Figure 12. Chromatogram data view with a list of 12 components

A combined scan for the MS1 scan stage appears on the Spectrum page of the MS spectrum view

(Figure 13).

Options menuFISh chromatogram

Figure 13. MS spectrum view open to the Spectrum page

2. To display the first MS2 scan for the selected component, click the MS2 node of the spectrum tree.

Figure 14. Spectrum page showing scan no. 3736 at full scale

On the Spectrum page, each FISh peak is color-coded:

The unexplained peaks are a royal blue .

• ( ) Explained Red indicates that the peak matches a specified fragment.

• ( ) Mass Diff./Modified Light blue indicates that the peak matches a shifted fragment for a

specified modification.

• ( )Neutral Loss Lime green indicates that the peak matches a specified fragment

from a neutral loss.

• ( ) Isotope explained peak Green indicates that the peak matches an isotopic ion for a

specified fragment.

Click to display the first MS2 scan

for the component.

The pink triangle marks the m/z value

of the precursor ion.

3. To zoom in on a specific peak, drag the pointer from left to right across a small range of m/z values on

the x axis.

This figure shows scan no. 3736 with the selection of the m/z 191–196 range on the x axis.

Figure 15. Scan no. 3736 showing the selection of the m/z 191–196 range on the x axis

4. To show the explanation for a FISh peak, point to the peak until the triangular marker appears

(Figure 16). Then, right-click and choose Show FISh Explanation m/z Va l u e (Figure 17).

Figure 16 shows the spectrum peak for m/z 195.0219 ( ). The peak is red, which indicates that its

m/z value matches one of the fragments in the user-specified fragments list (see card 8 in Figure 6).

Figure 16. Triangular marker that appears when you point to a peak

Figure 17. Spectrum shortcut menu

Zoom pointer

Triangular marker

Reset Zoom

icon

The FISh page opens in the spectrum view and displays the most likely explanation for the product

ion (Figure 18). The count number at the bottom of the page is the total number of explanations in

the list (with Show Base, Show Modification, and Show Neutral Loss turned on).

Figure 18. Parent (precursor ion) view on the FISh page

5. To view the fragment structure by itself, click Show Parent.

Figure 19. Fragment structure (product ion) view on the FISh page of the spectrum view

6. In the MS spectrum view, click the Spectrum tab to display the Spectrum page. Then, to reset the

zoom level, click the Reset Zoom icon, , to the right of the scan header (Figure 16).

7. To display the explanations for a shifted peak (a fragment with the specified modification), do the

following:

a. On the Spectrum page, zoom in on the m/z 188-218 range on the x axis.

b. Point to the light-blue peak at m/z 214.05342 until the triangular marker appears. Then,

right-click and choose Show FISh Explanation m/z 214.05342.

Note Clicking Show Parent toggles the displayed structure between these views:

• The parent structure (precursor ion) with the bonds for the fragment structure (product ion)

shown in red

• The fragment structure by itself

Figure 20 shows the “Show Parent” view on the left and the “fragment” view on the right for

m/z 214.05342 ( ).

Figure 20. FISh explanation for m/z 214.05342 (modification = oxidation)

8. To display the explanations for a neutral loss peak (a fragment produced by a neutral loss), do the

following:

a. On the Spectrum page, reset the zoom level, and then zoom in on the m/z 133-138 range on the

x axis.

b. Point to the lime green peak at m/z 136.07573 until the triangular marker appears. Then,

right-click and choose Show FISh Explanation m/z 136.07573.

Figure 21 shows the Show Parent view on the left and the fragment view on the right for

m/z 136.07573 ( ).

Figure 21. FISh explanation for m/z 136.07573 (fragment from a neutral loss)

Identify

detected

components

From the Chromatogram Processor module, you can run a library search to identify the detected

components. For this tutorial, run an Identity search against the mzCloud™ mass spectral database. This

online database contains entries for omeprazole (the analyte in this tutorial) and its impurities.

Note The application calculates neutral losses by subtracting the m/z value for a fragment structure

from the highest m/z value for a structure in the fragment list on the FISh Model page.

Parent structure Fragment structure

IMPORTANT To search the online mzCloud mass spectral database, your processing computer must be

connected to the Internet and have unblocked access to the mzCloud server.

YTo identify components by running a library search

1. (Optional) Run a connection check as follows:

a. From the application tab bar, click the Start tab to open the Start menu, and then click

Connection Check.

b. In the Connection Check dialog box, click Run.

The application verifies the connection.

c. If the connection is successful, go to the next step of this procedure.

d. If the application cannot connect to the mzCloud server, check the computer’s Internet

connection and its access to various sites. Also, make sure that the computer is synchronized with

Internet time.

2. In the Search group of the Chromatogram Processor toolbar, click Components Search.

The Component Search view opens to the right of the chromatogram and MS spectrum views.

3. In the Search Type list, select Identity.

4. In the Library list, select the Reference check box under mzCloud libraries.

5. To run a search for selected components, select the components in the chromatogram data view. For

this tutorial, select Component 5 in the Components list. Then, in the Component Search view,

click Search Selected.

If the search finds a matching precursor m/z and spectrum in the spectral library, the following items

appear:

• The match score and compound name appear in the Match column of the chromatogram data

view.

• The Spectra Compare page opens in the MS spectrum view.

• Information about the matching mzCloud entry appears in the Component Search view.

Figure 22 shows the search result for component 5 against the mzCloud Reference library.

Note Occasionally, the mzCloud Web site goes offline. When this happens, the mzCloud status

readback to the right of the application tab bar changes from Online to Offline (in red).

Note If you do not select a component, the application runs the search on component 1.

Figure 22. Search result for component 5

6. To run a search for all the detected components with MS2 scans, click Search All.

Figure 23 shows the result of an mzCloud identity search run in March 2023 against the mzCloud

Reference library. As new compounds are constantly being added to the mzCloud library, your results

might differ.

Figure 23. Search All results for the mzCloud Reference library

Compound’s match score

and name

Spectra Compare page

7. To search both the mzCloud Reference and Autoprocessed libraries, do the following in the

Component Search view:

a. Open the Library list.

b. Select the Reference and Autoprocessed check boxes.

c. Click Search All.

The result for component 4 changed because the search found a compound with a higher match score

in the Autoprocessed library.

Figure 24. Search All results for the mzCloud Reference and Autoprocessed libraries

Save the results

to an HCCX file

You can save the component detection and component annotation results to an HCCX file.

YTo save the results to an HCCX file

1. In the File group of the Chromatogram Processor toolbar, click Save, and then click Chromatogram

As.

2. Select a file location, name the file, and click Save.

Note The Reference library contains compounds with spectra that have been fully curated by mass

spectrometry specialists. The Autoprocessed library contains compounds with spectra that have

only been automatically processed with a curation software application.

Tip You can save the intermediate and final component detection and annotation results to

HCCX files so you can return to those results at a later time. Saving HCCX files lets you return to

a specific results state, and then perform different processing actions on the data.

Create and edit

a fragments list

In this tutorial, you used a predefined fragments list to create a FISh model for the analyte of interest. This

topic shows you how to create a fragments list with an appropriate set of fragments for the analyte of

interest in this tutorial—omeprazole.

Follow these topics in order:

1. create a fragments list and save it to an SDF file

2. Edit a fragments list

create a

fragments list

and save it to an

SDF file

YTo create a fragments list and save it to an SDF file

1. On the Spectrum page of the MS spectrum view, display the highest abundance MS2 scan for

omeprazole (precursor m/z 346) by selecting this scan from the MS2 Scans list in the chromatogram

data view.

Figure 25. Selecting scan no. 4266—the highest abundance MS2 scan for omeprazole

The spectrum appears on the Spectrum page (Figure 26 on page 21).

2. Right-click the spectrum and choose Copy MS Spectrum.

Note To create a fragments list, you can use the Batch Fragment Generation wizard or the

SledgeHammer module.

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Thermo Fisher Scientific MASS Owner's manual

Brand: Thermo Fisher Scientific

Model: MASS

Type: Owner's manual

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Thermo Fisher Scientific MASS Owner's manual

Thermo Fisher Scientific MASS Owner's manual

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